The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen.

Abstract

Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50ng/ml) and macrophage colony stimulating factor (50ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9days for spleen, as compared to 5days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm(2) for spleen, as compared to 50,000/cm(2) for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70-80% of cells were lost during the first week of freezing, during the subsequent 5weeks the losses were within 2-5% per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics.